Analyte Accumulation in Hydrogel Microparticles - Fabricating the Perfect Particle

Introduction: : Collections of hydrogel microparticles serve not only as tissue culture scaffolds or drug delivery vehicles but also play crucial roles in lab-on-a-particle technologies, enabling miniaturized, compartmentalized assays for single-cell analysis and low-copy-number diagnostics. Hydrogels have highly tunable structural properties, which can be tailored through functional groups, porogens, polymer precursors, and fabrication methods. These factors directly influence properties including stiffness, biocompatibility, and permeability. Hydrogel microparticles offer distinct advantages over bulk hydrogels such as higher surface area-to-volume ratios, enhanced malleability, and improved compartmentalization capabilities.
By facilitating multiple independent reactions, populations of microparticles statistically improve assay accuracy and enable multiplexing capabilities. Despite their widespread applications, no systematic study has investigated how hydrogel properties affect analyte uptake based on molecular weight. Most research has focused on small molecules, leaving significant knowledge gaps regarding the upper size limit of analytes that hydrogels can accumulate. This study examines how polymer molecular weight, polymer concentration, and UV crosslinking intensity influence signal accumulation in hydrogel microspheres—findings that have potential applications in highly sensitive diagnostic assays and colorimetric microparticle-based detection.

Materials and

Methods: : To efficiently fabricate diverse hydrogel microparticles while minimizing device transitions, we employed a flow-focusing microfluidic droplet generator with two inlets. One inlet delivered a 25-weight percent PEG solution, while the other supplied a PEG-free diluent. By adjusting the flow rates, we precisely controlled the hydrogel microsphere weight percent, enabling production of multiple different hydrogel variations in a single manufacturing session and significantly reducing fabrication time. We generated 36 distinct hydrogel particle formulations with systematically varied molecular arm length, polymer weight percent, and UV crosslinking intensity. We then investigated signal accumulation in these particles through incubation with three classes of analytes: oligonucleotides, fluorophores, and gold nanoparticles.
For each analyte class, we selected three representative molecules of increasing size: oligonucleotides (30, 45, and 60 base pairs), fluorophores (~60 kDa, ~165 kDa, and ~300 kDa), and gold nanoparticles (5 nm, 10 nm, and 20 nm). Accumulation over time was achieved due to engineered affinity with the hydrogel. For fluorophore and gold nanoparticle assays, molecules and microparticles were incubated together for one week before excess molecules were removed by washing. We then acquired images and measured either fluorescence intensity or color saturation, depending on the analyte. For oligonucleotide experiments, we first incubated hydrogel microparticles with oligos of different lengths for one week. Subsequently, we performed a second one-week incubation with complementary fluorophore-labeled oligos. Fluorescence images were then acquired for these microparticles as well. All results were normalized by particle size to enable direct comparison across formulations.

Results, Conclusions, and Discussions:: Our findings demonstrate that hydrogel microparticles with the longest arm lengths and lowest weight percent generally exhibited the highest signal intensity across different analyte types. For fluorophores, we observed an average 10-fold increase in fluorescence with decreasing weight percent and a 55-fold increase with increasing arm length. Similarly, for gold nanoparticles, microparticles with the longest molecular arms and lowest weight percent were the only formulations showing colorimetric signal across all nanoparticle sizes. This trend of increasing signal with increasing arm length and decreasing molecular weight was maintained for smaller gold nanoparticles.
We anticipated that oligonucleotides would follow patterns similar to fluorophores, given their shared binding moiety and comparable fluorophore size. However, we observed that the "tightest mesh" microparticles (those with the lowest arm length and highest weight percent) consistently demonstrated the lowest fluorescence intensity, suggesting significant barriers to molecular penetration in these specific hydrogel formulations. Notably, fluorescence intensity increased at least 2-fold, and on average 7-fold, when moving from these tight mesh microparticles to formulations with either slightly larger arm length or lower weight percent. Beyond these threshold adjustments, fluorescence intensity remained within two standard deviations across formulations. We hypothesize that accumulation of negative charge from increasing numbers of oligonucleotides and longer base pairs may have inhibited entry of complementary fluorophore-labeled oligos, explaining the general decrease in fluorescence as base pair length increased.
This study identifies polymer molecular weight and polymer concentration as key determinants of signal accumulation in hydrogel microparticles. Our findings demonstrate that particles manufactured with 7.5 weight percent and 40kDa arm length PEG can successfully accumulate colorimetric signal from gold nanoparticles up to 20nm in size. These insights can inform modifications to hydrogel properties for improved therapeutic transport in tissue engineering applications. Furthermore, the enhancement in signal accumulation can increase the sensitivity of hydrogel microparticle-based lab-on-a-particle detection assays and expand their dynamic range.

Acknowledgements and/or References (Optional):: This work was funded by the NSF PATHS-UP Engineering Research Center #164845.